MOLECULAR SYSTEMATICS OF THE GENUS Dichaea (ZYGOPETALINAE: ORCHIDACEAE) By KURT MAXIMILLIAN NEUBIG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005 Copyright 2005 by Kurt Maximillian Neubig This thesis is dedicated to all people of planet Earth. ACKNOWLEDGMENTS I would like to thank my advisor, Norris H. Williams, for his guidance. He also chose my thesis topic and it was an excellent one. I would also like to thank W. Mark Whitten, one of the smartest men I have ever met. Both Norris and Mark have been invaluable contributors to this project and to my education. Walter Judd has been an uncontainable source of enthusiasm for everything botanical. He has also been a generous member of my committee. Franco Pupulin of Lankester Gardens in Costa Rica contributed many vouchered specimens and DNA sources from Costa Rica for this study. His insights into the quirks of taxonomy in Dichaea have been very enlightening. I would like to acknowledge the remarkable generosity of the Portillas of Ecuagenera in Cuenca, Ecuador, who have been most helpful with their amazing collection of Ecuadorian orchids. Also, Bruce Holst allowed me to sample several dozen herbarium specimens for DNA at Marie Selby Botanical Gardens. Mario Blanco has been willing to collect Dichaeas whenever he has been in the field. Barbara Sue Carlsward helped me in so many different ways. She gave me unfathomable insight into computer software, advice about graduate student life in general, and has been a good friend. I would also like to thank all the other graduate students and faculty at the University of Florida who have enhanced my education. iv Robert Dressler has always been willing to help me identify specimens and has always been willing to admit how befuddling they are. Calaway Dodson has on several occasions been willing to empathize with me on the difficulty and problems of Dichaea taxonomy. Gustavo Romero has generously spent his time to help me with a handful of citations. The Lewis and Varina Vaughn Fellowship in Orchid Biology and the American Orchid Society’s 11th World Orchid Conference Fellowship supported this research. In addition, much of the cost of lab work was supported by the generosity of Norris Williams and Mark Whitten. I am grateful to my parents, Henry and Linda Neubig, who have doted upon me my entire life and who have always supported my poor choices (e.g., orchid systematics). I would also like to thank my wife, Julie Kay Neubig, who is perhaps the sweetest person I have ever met. She has been patient and very supportive. She is perfect in every way. v TABLE OF CONTENTS page ACKNOWLEDGMENTS .......................................................................................iv LIST OF TABLES ............................................................................................... viii LIST OF FIGURES ...............................................................................................ix ABSTRACT ..........................................................................................................xi CHAPTER 1 INTRODUCTION............................................................................................ 1 Taxonomic history.......................................................................................... 9 Morphology .................................................................................................. 12 Anatomy....................................................................................................... 15 Karyology ..................................................................................................... 16 Distribution ................................................................................................... 17 Pollination biology ........................................................................................ 18 2 MATERIALS AND METHODS ..................................................................... 21 Plant Material ............................................................................................... 21 Extraction ..................................................................................................... 23 Amplification................................................................................................. 24 Sequencing .................................................................................................. 27 Data Analysis ............................................................................................... 28 3 RESULTS .................................................................................................... 31 ITS Analyses of Dichaea.............................................................................. 31 matK Analyses of Dichaea ........................................................................... 34 trnL-F Analyses of Dichaea.......................................................................... 37 Combined Plastid Analyses of Dichaea ....................................................... 40 Combined ITS and Plastid Analyses of Dichaea.......................................... 43 Analysis of a Hybrid Accession of Dichaea schlechteri ................................ 46 4 DISCUSSION............................................................................................... 49 Section Dichaeopsis..................................................................................... 50 vi Section Pseudodichaea ............................................................................... 54 Section Dichaea (Including Section Dichaeastrum) ..................................... 56 Summary...................................................................................................... 61 LIST OF REFERENCES .................................................................................... 63 BIOGRAPHICAL SKETCH ................................................................................. 68 vii LIST OF TABLES Table page 1-1 Taxonomic history of Dichaea and the sections of Dichaea....................... 11 2-1 Taxa used for molecular phylogenetic study.............................................. 21 2-2 Primer sequences for polymerase chain reaction. ..................................... 26 2-3 Components of polymerase chain reactions. ............................................. 26 2-4 Thermocycler programs for polymerase chain reaction. ............................ 27 2-5 Thermocycler program for cycle sequencing. ............................................ 28 2-6 Cycle sequencing reagents........................................................................ 28 3-1 Comparison of tree statistics for each gene region and combinations of these gene regions for parsimony analyses............................................... 31 viii LIST OF FIGURES Figure page 1-1 Photos of flowers and plants vouchered in this study. ................................. 2 1-2 Photos of flowers and plants vouchered in this study. ................................. 3 1-3 Photos of flowers and plants vouchered in this study. ................................. 4 1-4 Photos of flowers and plants vouchered in this study. ................................. 5 1-5 Photos of flowers and plants vouchered in this study. ................................. 6 1-6 Photos of flowers and plants vouchered in this study. ................................. 7 1-7 Tree modified from Whitten et al. (2005) showing relationships in the Zygopetalinae based on ITS, matK, and trnL-F. .......................................... 8 1-8 Figure modified from Folsom (1987) indicating the hypothesized relationships of species within Dichaea section Dichaea. .......................... 12 1-9 Photos of various aspects of morphology in Dichaea. ............................... 13 1-10 Distribution of the genus Dichaea. ............................................................. 18 2-1 Diagrams of selected gene regions (ITS, matK, and trnL-F) used in this study.. ........................................................................................................ 25 3-1 One of 503 equally parsimonious ITS trees. .............................................. 32 3-2 Jackknife consensus tree from analysis of ITS data set ............................ 33 3-3 One of 5991 equally parsimonious matK trees. ......................................... 35 3-4 Jackknife consensus tree from analysis of matK data set.......................... 36 3-5 One of >20,000 equally parsimonious trnL-F trees .................................... 38 3-6 Jackknife consensus tree from analysis of trnL-F data set. ....................... 39 3-7 One of >20,000 equally parsimonious plastid (matK and trnL-F) combined trees........................................................................................................... 41 ix 3-8 Jackknife consensus tree from analysis of plastid (matK and trnL-F) data set. ............................................................................................................. 42 3-9 One of >20,000 equally parsimonious three-gene (ITS, matK, and trnL-F) combined trees .......................................................................................... 44 3-10 Jackknife consensus tree from analysis of three-gene (ITS, matK, and trnL- F) data set.................................................................................................. 45 3-11 50% majority-rule